We propose to develop an in vivo reporter system that will specifically monitor activation of NF-?B pathways in vivo in disease states. In general, NF-?B transcription factors represent an important, but complex, set of therapeutic targets for many diseases, including inflammatory arthritis and cancer. NF-?B uses two distinct routes of activation, the classical and alternative pathways. In the classical pathway, ligand-induced activation of IKK2 leads to degradation of I?B1. The controlled step in the alternative pathway is the partial degradation of the I?B protein p100, initiated by the kinase NIK. Classical NF-?B signaling is ubiquitous in normal physiology, complicating its targeting in disease states. Alternative NF-:B activation appears to be much more restricted, providing potential benefits for drug targeting. We have shown that mice lacking NIK are resistant to both lymphocyte-mediated initiation of arthritis, and to bone resorption associated with inflammatory arthritis, thus emphasizing the importance of the alternative NF-?B activation pathway during the disease response. We hypothesize that, since the degradation of I?B1 and p100 represent the principal regulated steps in activation of the classical and alternative NF-?B pathways, respectively, reporters monitoring these events will be powerful tools for the study of NF-?B signaling in the context of arthritis, and for evaluating drugs targeting NF-?B. To this end, we will fuse VP16-Gal4BD (VG), a potent transcriptional activator, to the N-terminus of either p65 or p100. In resting cells the reporter constructs will be retained in the cytoplasm by the normal control mechanisms for p65 and p100. Upon pathway activation such as with cytokine stimulation, the reporters will travel to the nucleus where the VG component will transactivate a Gal4-response element driven Fluc gene. In this system, the Fluc signal is proportional to pathway activation, with a broad dynamic range. Aim 1: Generation and validation of p100-processing and I?B1-degradation dependent reporter constructs in vitro. Aim 2: Generation of VG-p100 and VG-p65 reporter mice and analysis of alternative and classical NF-?B signaling during inflammatory arthritis in vivo. PUBLIC HEALTH RELEVANCE: These studies provide a novel use of real-time 2-color bioluminescence imaging to non-invasively monitor NF-?B pathways known to be important in inflammatory diseases such as arthritis. Individuals can be imaged multiple times, generating a new dynamic picture of NF-?B activation in the context of disease, and to understand how various interventions modify disease progression in molecular terms, in vivo and in real time. This will represent a major advance in understanding the mechanisms of both the pathophysiology of disease and drug actions. [unreadable] [unreadable] [unreadable]